Mol Imaging Biol (2014) 16:322Y329
DOI: 10.1007/s11307-013-0693-0* World Molecular Imaging Society, 2013
Published Online: 18 October 2013

RESEARCH ARTICLE

Differences in Transport Mechanisms of trans-1Amino-3-[18F]Fluorocyclobutanecarboxylic Acid
in Inflammation, Prostate Cancer, and Glioma
Cells: Comparison with L-[Methyl-11C]Methionineand 2-Deoxy-2-[18F]Fluoro-D-Glucose
Shuntaro Oka,1,5 Hiroyuki Okudaira,1 Masahiro Ono,1,2 David M. Schuster,3Mark M. Goodman,3 Keiichi Kawai,2,4 Yoshifumi Shirakami1
1

Research Center, Nihon Medi-Physics Co., Ltd, Chiba, Japan
Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan
3
Division of Nuclear Medicine and Molecular Imaging, Department of Radiology and Imaging Sciences, Emory University, Atlanta, GA, USA
4
Biomedical Imaging Research Center, University of Fukui, Fukui, Japan
5
Kitasode 3-1, Sodegaura, Chiba, 299-0266, Japan
2

Abstract
Purpose: We aimed to elucidate trans-1-amino-3-[18F]ﬂuorocyclobutanecarboxylic acid (anti[18F]FACBC) uptake mechanisms in inﬂammatory and tumor cells, in comparison with those of
11
11
18
18
L-[methyl- C]methionine ([ C]Met) and 2-deoxy-2-[ F]ﬂuoro-D-glucose ([ F]FDG).
Procedures: Using carbon-14-labeled tracers, in vitro time-course, pH dependence, andcompetitive inhibition uptake experiments were performed in rat inﬂammatory (T cells, B cells,
granulocytes, macrophages), prostate cancer (MLLB2), and glioma (C6) cells.
Results: Anti-[14C]FACBC uptake ratios of T/B cells to tumor cells were comparable, while thoseof granulocytes/macrophages to tumor cells were lower than those for [14C]FDG. Over half ofanti-[14C]FACBC uptake by T/B and tumor cells was mediated by Na+-dependent amino acidtransporters (system ASC), whereas most [14C]Met transport in all cells was mediated by Na+independent carriers (system L).
Conclusions: The low anti-[18F]FACBC accumulation in granulocytes/macrophages may be
advantageous in discriminating inﬂamed regions from tumors. The signiﬁcant anti-[18F]FACBC
uptake in T/B cells may cause false-positives in some cancer patients who undergo FACBCpositron emission tomography (PET).
Key words: Anti-[18F]FACBC, Anti-[14C]FACBC, [11C]Met, [14C]Met, [18F]FDG, [14C]FDG, Aminoacid transporters, Inﬂammatory cells, Prostate cancer, Glioma

Electronic supplementary material The online version of this article(doi:10.1007/s11307-013-0693-0) contains supplementary material, whichis available to authorized users.
Correspondence to: Shuntaro Oka; e-mail: shuntaro_oka@nmp.co.jp

Introduction

2-D

eoxy-2-[18F]ﬂuoro-D-glucose ([18F]FDG) is themost frequently used positron emission tomog-

S. Oka et al.: Anti-[18F]FACBC Transport Mechanisms in Inflammatory and Cancer Cells

raphy (PET) tracer for cancer imaging; however, it accumulates
in inﬂammatory regions, potentially resulting in false-positive
uptake [1]. Alternatively, L-[methyl-11C]methionine ([11C]Met),an amino acid (AA) PET tracer, is useful for tumor imaging andmonitoring of therapeutic effects, especially in brain, due to its
low physiological accumulation in normal cerebral tissue andinﬂamed lesions [2]. However, its use is restricted to facilities
with a cyclotron, because carbon-11 has a short half-life(20 min). To overcome this, several synthetic ﬂuorine-18-labeled
AA PET tracers (half-life: 110 min) such as trans-1-amino-3[18F]ﬂuorocyclobutanecarboxylic acid (anti-[18F]FACBC) havebeen developed [3]. We and others have reported that theimaging potential of anti-[18F]FACBC for glioma and prostatecancer (PCa) is comparable to that of [18F]FDG and [11C]Metin preclinical and clinical studies [3–6]. Moreover, wepreviously reported that anti-[18F]FACBC accumulation was
higher in subcutaneous PCa than in lymphadenitis in an animalmodel [5]. However, a recent clinical study showed no
separation between malignant and non-malignant sextants bystandardized uptake value (SUV) on FACBC-PET in somePCa patients [7]. We speculated that the relatively high SUV in
non-malignant sextants might be explained by anti[18F]FACBC uptake and accumulation in inﬂammatory cells,
because PCa usually contains inﬂamed tissue [8].
Transport of anti-[18F]FACBC into cells is mediated by AAtransporter(s) (AAT(s)) [9–11], which are either Na+-dependentor Na+-independent, based on the sodium ion requirement fortransport [12]. AATs are further categorized into "systems" (e.g.,system A, ASC, L) based on their substrate selectivity [12].
Moreover, extracellular pH affects AAT activity [12]. We
previously reported that the main AATs mediating trans-1amino-3-ﬂuoro[1- 14C]cyclobutanecarboxylic acid (anti[14C]FACBC) uptake in human PCa cell lines were Na+dependent system ASC (especially ASCT2) and Na+-independent system L (especially LAT1), and the contribution of system
L was enhanced in acidic pH as is present in an intratumoral
environment [9–11].
Although anti-[18F]FACBC transport mechanisms in cancer
cells have been demonstrated, transport in inﬂammatory cells
remains unclear. Therefore, we compared anti-[18F]FACBC
uptake in rat inﬂammatory cells (T cells, B cells, granulocytes,
macrophages) with that in rat PCa (MLLB2) and glioma (C6)cell lines, and also to that of [18F]FDG and [11C]Met.

Materials and Methods

323

American Radiolabeled Chemicals (St. Louis, MO, USA), withspeciﬁc activities of 2.04 and 11.10 GBq/mmol, respectively.

Animals and Cells
All animal handling and experimentation procedures accorded with
protocols approved by the committee on animal welfare at Nihon
Medi-Physics. Male Copenhagen rats (CLEA, Tokyo, Japan) wereused in experiments at 11–16 weeks of age. In all animal preparations,animals were anesthetized with 1 % isoﬂurane (Mylan, Pittsburgh, PA,
USA).
Inﬂammatory cells were isolated from Copenhagen rats using
standard methods based on density gradient centrifugation (T cells, B
cells, and granulocytes) and adhesion-mediated puriﬁcation on culture
dishes (macrophages). T cells and granulocytes were stimulated withconcanavalin A (0.25 μg/ml) and phorbol 12-myristate 13-acetate(100 nM), respectively; B cells and macrophages were stimulated withlipopolysaccharide (5 μg/ml) from Escherichia coli O55:B5. See
Supplemental Information for details.
Rat PCa (MLLB2) and glioma (C6) cell lines were obtained from
American Type Culture Collection (ATCC, Manassas, VA, USA), andmaintained in RPMI1640 and modiﬁed Eagle medium, respectively,
supplemented with 10 % fetal bovine serum (ATCC), 100 μg/ml
streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine.

Time-Course Tracer Uptake Experiments
Time-course tracer uptake experiments were carried out based on
previous studies [5, 10] using 10 μM anti-[14C]FACBC, [14C]Met,and [14C]FDG. In these experiments, we used glucose-free Hanks'balanced salt solution containing Mg2+ and Ca2+ (HBSS(+)) because
145
D-glucose inhibits [ C]FDG uptake. Brieﬂy, T/B cells (5.0×10 ) or
granulocytes (2.5×105) were suspended in 100 μl glucose-free
HBSS(+) including tracers, and placed in 96-well culture plates. For
macrophages, 150 μl of glucose-free HBSS(+) containing tracers wasadded to macrophages cultivated in 48-well culture plates (3.0×105/
well). MLLB2 (5.0×104/well) and C6 (1.0×105/well) cells wereseeded in 24-well culture plates, and media were replaced with 300 μl
glucose-free HBSS(+) containing tracers on day 2 of cultivation. Cells
were incubated for 5, 15, 30, and 60 min at 37 °C, washed with icecold glucose-free HBSS(+), and lysed in 0.1 N NaOH. Radioactivity
in cell lysates was measured with a Tri-Carb 2910TR liquid
scintillation counter (Perkin Elmer, Waltham, MA, USA). Protein
concentrations of cell lysates were determined using the BCA Protein
Assay Kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA). Tracer
uptake was expressed as μmol/mg protein.

Chemicals
pH-Dependent Tracer Uptake Experiments
All reagents were purchased from Life Technologies (Carlsbad,
CA, USA), Sigma-Aldrich (St. Louis, MO, USA), Nacalai Tesque(Kyoto, Japan), and Wako (Osaka, Japan), unless otherwise stated.
In all experiments, carbon-14-labeled compounds were used as
tracers. Anti-[14C]FACBC was synthesized by Sekisui Medical(Tokyo, Japan) as described previously [10], with speciﬁc activity
and radiochemical purity of 2.08 GBq/mmol and 998.0 %,respectively. L-[Methyl-14C]methionine ([14C]MET) and 2-ﬂuoro2-deoxy-D-glucose, [14C(U)] ([14C]FDG) were purchased from

Anti-[14C]FACBC and [14C]Met uptake experiments were conductedusing activated T/B cells and tumor cells according to the proceduredescribed above. Three types of uptake buffer, sodium, choline, andlithium, were used, because 1) all AATs function in sodium buffer, 2)
only Na+-independent AATs function in choline buffer, and 3) all Na+independent AATs and the Na+-dependent N and y+L AAT systems
function in lithium buffer [12, 13]. The sodium buffer contained
140 mM NaCl, 5 mM KCl, 5.6 mM D-glucose, 0.9 mM CaCl2,

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S. Oka et al.: Anti-[18F]FACBC Transport Mechanisms in Inflammatory and Cancer Cells

1.0 mM MgCl2, 0.3 mM Na2HPO4, and 0.4 mM KH2PO4; pH wasadjusted with 4-morpholineethanesulfonic acid to pH 6.0 and with 4(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) to pH 7.0
and 8.0. The choline buffer contained 140 mM choline chloride and
0.3 mM K2HPO4 instead of sodium chloride and Na2HPO4. The
lithium buffer contained lithium chloride instead of choline chloride.
Tracer uptake times were 5 min for MLLB2 and C6 and 10 min for T/
B cells, because anti-[14C]FACBC uptake in each cell type was linear
up to these time-points (data not shown). Termination of tracer uptake,cell washing, and radioactivity and cellular protein measurements
were conducted as described above. Tracer uptake was expressed as
pmol/mg protein/min. The contributions of Na+-dependent and Na+independent transport systems to anti-[14C]FACBC and [14C]Metuptake were estimated using the following formulas:Naþ ‐independent AATs ¼ V C
Naþ‐dependent AATs ¼ V S −V C
system N and yþ L AATs ¼ V L −V C
Naþ ‐dependent AATs except for system N and yþ L AATs ¼ V S −V L ;where VS, VC, and VL are the amounts of tracer uptake in sodium,choline, and lithium buffers, respectively. The transport of tracers in
sodium buffer at each pH was normalized to 100 %. If tracer uptake
in choline buffer was higher than that in sodium buffer at each pH,
the contribution of Na+-dependent AATs was taken as zero. If tracer
uptake in lithium buffer was higher than that in sodium buffer at
each pH, the uptake in lithium buffer was considered the same as in
sodium buffer.

Competitive Inhibition Tracer Uptake Experiments
Sodium, choline, and lithium buffers, adjusted to pH 7.2–7.3 withHEPES, were used. Uptake experiments were performed as describedabove in the presence of 2 mM naturally occurring or synthetic AAs as
inhibitors. The synthetic AAs and their AAT speciﬁcities are(summarized in Table S1): 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), systems L, B0, and B0,+; N-ethylmaleimide (NEM),
systems L (except LAT1), b0,+, and y+; and 2-(methylamino)isobutyric acid (MeAIB), systems A, IMINO, and PAT. Naturally
occurring AAs are not AAT-speciﬁc, but some show preference forcertain AATs; thus, we used some naturally occurring AAs (L-forms)
as inhibitors, as follows (preferred AATs in parentheses): glutamine,systems A, ASC (ASCT2), B0 (B0AT1), N, y+L, L (LAT2), and B0,+;
serine, systems A, ASC, B0 (B0AT1), N (SNAT5), L (LAT2), asc, and
B0,+; phenylalanine, systems B0, y+L, L, T, B0,+, and b0,+; proline,
system A (ATA2), ASC (ASCT1), G-like, B0, IMINO, B0,+, and PAT;
glycine, systems A (except for ATA1), B0 (B0AT1), N (SNAT5), y+L,−
L (LAT2), asc, B0,+, and, PAT; glutamate, systems y+L, XA,G, and
0,+
0,+
+
+
14
−
XC; arginine, systems B , b , y L, and y . Anti-[ C]FACBC and[14C]Met transport (control) in the absence of inhibitors in each buffer
was normalized to 100 %, and the inhibitory effects of naturally
occurring and synthetic AAs on the uptake of both tracers were
calculated as a percentage of control. To simplify the results, theintensity of the inhibitory effects was categorized into ﬁve classes
based on average inhibitory rates: ≤19.9 %, 20.0–39.9 %, 40.0–
59.9 %, 60.0–79.9 %, and ≥80.0 %.

Statistics
Experiments were repeated at least twice. All results are expressed
as mean±SD unless otherwise stated. All statistical analyses were
performed using SAS for Windows (Ver. 5, SAS Institute, Cary,
NC, USA). For datasets with normal distribution, homogeneity ofvariance was analyzed by the F-test; homogeneous data wereanalyzed using the two-tailed unpaired Student's t-test, whereas
non-homogeneous data were analyzed using the Welch t-test. The
Wilcoxon rank sum test was used for non-normal datasets. PG0.05
was considered signiﬁcant.

Results
Time-Course Tracer Uptake Experiments
The time-course of anti-[14C]FACBC, [14C]Met, and [14C]FDG
uptake was determined in inﬂammatory cells and tumor cells(Fig. 1); thereafter, uptake ratios of activated inﬂammatory cells
to non-stimulated (NS) inﬂammatory cells and of activatedinﬂammatory cells to tumor cells were calculated from theresults in Fig. 1 (Table 1). Although anti-[14C]FACBC uptake
in all cells except granulocytes tended to plateau at 15 or 30 minof incubation, [14C]Met and [14C]FDG uptake increased withtime (Fig. 1). [14C]FDG uptake by activated inﬂammatory cells
and tumor cells was signiﬁcantly higher than those of AA
tracers (Fig. 1, note difference in Y-axis scale between tracers).
Upon inﬂammatory cell activation, anti-[14C]FACBC uptake
tended to be accelerated except in macrophages (Fig. 1a,b,
Table 1), whereas [14C]Met uptake was increased in T/B cells,
but not in granulocytes/macrophages (Fig. 1c,d, Table 1). All
activated cells took up more [14C]FDG than did non-stimulated
cells (Fig. 1e,f, Table 1).
Anti-[14C]FACBC uptake was similar in activated T/Bcells and MLLB2/C6 cells, but clearly lower in activatedgranulocytes/macrophages (Fig. 1a,b). [14C]Met uptake inactivated T cells and [14C]FDG uptake in all activated cells
except B cells were similar to that in tumor cells (Fig. 1c–f).
Uptake ratios of activated inﬂammatory cells to tumor cells
show the relatively high uptake of anti-[14C]FACBC in
activated T/B cells (Table 1).

pH-Dependent Tracer Uptake Experiments
The transport activities of some AATs are affected by extracellular pH changes. Some examples of such pH-dependent activity
changes are given in Table S1. Thus, we investigated theinﬂuence of extracellular pH on anti-[14C]FACBC and [14C]Metuptake in activated T/B cells and tumor cells, which indicatedrelatively high AA tracer uptake.
[14C]Met uptake tended to be higher in acidic pH in allconditions (Fig. 2a–d). Although similar anti-[14C]FACBC
uptake trends were observed in the absence of Na+, anti[14C]FACBC uptake in sodium buffer was higher at neutral
and acidic pH than at alkaline pH (Fig. 2a–d).

S. Oka et al.: Anti-[18F]FACBC Transport Mechanisms in Inflammatory and Cancer Cells

Fig. 1. Time-course of anti-[14C]FACBC (a, b), [14C]Met (c,d), and [14C]FDG (e, f) uptake in non-stimulated (NS) (a, c, e)
and activated (b, d, f) rat T cells, B cells, granulocytes,
macrophages, and rat-derived PCa (MLLB2) and glioma (C6)cell lines (n=6–12). Statistical analyses were conducted
between each type of inflammatory cells and MLLB2 or C6.
*PG0.05 vs. MLLB2, †PG0.01 vs. MLLB2, ‡PG0.05 vs. C6,§
PG0.01 vs. C6.

325

Na+-dependent anti-[14C]FACBC transport tended to be
more active in neutral to alkaline pH, whereas Na+-independenttransport tended to be more active in acidic pH (Fig. 2e–h). The
contributions of Na+-dependent AATs except system N and y+L
to anti-[14C]FACBC uptake in T/B cells were 70–90 %, while
50–100 % of [14C]Met transport was mediated in an Na+independent manner (Fig. 2e,f). In tumor cells, although anti[14C]FACBC transport was predominantly Na+-independent inacidic pH (60–70 %), the contribution of Na+-dependent uptake,except in system N and/or y+L, was enhanced up to 60 % at
pH 8.0 (Fig. 2g,h). Almost all [14C]Met transport in tumor cells
was Na+-independent. The contributions of system N and/or y+L
to anti-[14C]FACBC and [14C]Met uptake were 10 % at most in
T/B cells and tumor cells.
As described above, time-course experiments of anti[14C]FACBC, [14C]Met, and [14C]FDG uptake were conductedin the absence of D-glucose to avoid [14C]FDG uptake inhibitionby D-glucose. However, because D-glucose is an important fuelfor cells, utilized at a high rate by inﬂammatory cells during
activation and by cancer cells, uptake ratios of activatedinﬂammatory cells to tumor cells in the presence of D-glucose
were calculated from the data in Fig. 2a–d. Anti-[14C]FACBC
and [14C]Met uptake ratios at pH 7.0 (Table 2), were lower thanthose in Table 1, indicating that although the inﬂammatory cellto tumor cell uptake ratios of anti-[14C]FACBC and [14C]Met
are improved under physiological conditions, anti-[14C]FACBC
ratios in activated T/B cells remain consistently higher than otherratios. Extracellular pH changes had little effect on these ratios.

Competitive Inhibition Tracer Uptake Experiments
To narrow down AATs involved in anti-[14C]FACBC and[14C]Met uptake, we investigated their uptake in the presence of
naturally occurring/synthetic AAs (Fig. 3, Figs. S2–S5). In

Table 1. Uptake ratio of activated inﬂammatory cells to non-stimulated inﬂammatory cells, and of activated inﬂammatory cells to tumor cells(without D-glucose)
Activated inﬂammatory
cells

T cells

B cells

Granulocytes

Macrophages

Incubation time
(min)

5
15
30
60
5
15
30
60
5
15
30
60
5
15
30
60

Anti-[14C]FACBC[14C]Met[14C]FDG

vs. NSvs. MLLB2

vs. C6

vs. NSvs. MLLB2

vs. C6

vs. NSvs. MLLB2

vs. C6

3.0
2.1
1.8
1.4
1.5
1.4
1.3
1.2
1.5
2.5
5.4
4.9
1.1
1.1
1.1
1.5

1.2
1.1
1.1
1.0
1.2
1.2
1.3
1.3
0.04
0.1
0.4
0.6
0.2
0.1
0.2
0.2

0.9
0.8
0.9
0.8
0.9
1.0
1.1
1.1
0.02
0.1
0.3
0.5
0.1
0.1
0.1
0.2

3.1
2.9
2.9
3.2
2.3
2.0
1.8
1.9
1.1
1.0
1.7
1.3
0.9
0.9
1.0
1.2

0.6
0.6
0.9
1.2
0.3
0.4
0.5
0.7
0.02
0.1
0.1
0.2
0.1
0.2
0.2
0.4

0.7
0.7
0.8
0.9
0.4
0.4
0.4
0.5
0.02
0.1
0.1
0.1
0.1
0.2
0.2
0.3

4.6
4.1
4.4
9.2
2.0
2.1
2.2
2.4
6.2
7.2
7.2
6.3
3.5
3.9
3.5
3.9

1.2
0.9
0.8
0.8
0.5
0.4
0.4
0.4
1.9
1.5
1.5
1.5
0.9
0.9
0.7
0.8

1.2
0.8
0.8
0.8
0.5
0.4
0.4
0.4
1.9
1.4
1.4
1.5
1.0
0.8
0.7
0.8

These ratios were calculated from Fig. 1. NS: non-stimulated

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S. Oka et al.: Anti-[18F]FACBC Transport Mechanisms in Inflammatory and Cancer Cells

Fig. 2. pH-dependence of anti-[14C]FACBC and [14C]Metuptake, and contributions of Na+-dependent and Na+independent transport systems to the uptake of AA tracers into
rat activated T cells (a, e), B cells (b, f), and PCa (MLLB2) (c, g)
and glioma (C6) (d, h) cell lines. a–d Cells were incubated with
10 μM anti-[14C]FACBC (FACBC) or [14C]Met (Met) in sodium,choline, or lithium buffer (n=6). *PG0.05, **PG0.01 relative toanti-[14C]FACBC uptake values at pH 7.0 in sodium, choline,
and lithium buffer. e–h Contributions of Na+-independent
uptake, uptake via systems N and/or y+L, and Na+-dependent
uptake except for systems N and y+L to anti-[14C]FACBC and[14C]Met uptake were calculated from the data in a–d.

sodium buffer, small neutral AAs such as glutamine and serine,
which are preferentially transported by systems A, ASC, B0(B0AT1), N, y+L, L (LAT2), asc, and/or B0,+, inhibited ≥80 %anti-[14C]FACBC uptake by T/B cells. On the contrary,

inhibition by glutamate, arginine, and MeAIB, which are−, XC−, A, IMINO,
substrates for systems B0,+, b0,+, y+L, y+, XA,G
and/or PAT, was ≤19.9 %. These results suggest thatsystems ASC, B0, N, L, and/or asc are the important
AATs that mediate anti-[14C]FACBC uptake in activated
T/B cells. Inhibition by phenylalanine and BCH, substrates ofsystems B0, y+L, T, L, B0,+, and/or b0,+, on anti-[14C]FACBCtransport by T/B cells was 20.0–59.9 % in the presence of
Na+, but ≥80.0 % in choline buffer (Fig. 3a,b). Althoughglutamine in the lithium buffer showed ≥80.0 % inhibitory
effect for anti-[14C]FACBC uptake in T/B cells (Fig. 3c),
this does not imply anti-[14C]FACBC transport via system
N AATs, because glutamine inhibited the transport≥80.0 % even in choline buffer (Fig. 3b). These results
indicate that system ASC is the main AAT among Na+dependent AATs for anti-[14C]FACBC transport in T/Bcells (Figs. 2e,f and 3). System L is also involved in Na+independent anti-[14C]FACBC transport, although its
contribution was 20 % at most (Fig. 2e,f).
System L is thought to be the main AAT for [14C]Metuptake in T/B cells, because most [14C]Met transport wasmediated in an Na+-independent manner (Fig. 2e,f) and therepresentative substrates for system L such as phenylalanine
and BCH showed strong inhibitory effects in choline buffer(Fig. 3b).
In neutral conditions, Na+-dependent and Na+-independentanti-[14C]FACBC transport had similar contributions in MLLB2
and C6 cells (Fig. 2g,h). Anti-[14C]FACBC inhibition by
naturally occurring/synthetic AAs was also similar between
both tumor cell lines (Fig. 3). In the presence of Na+, glutamineand serine showed the strongest inhibition of anti-[14C]FACBC
uptake, followed by phenylalanine, proline, glycine, and BCH.
Inhibitory rates by glutamate, arginine, NEM, and MeAIB were
≤19.9 %. Removal of Na+ from the buffer led to ≥80.0 %inhibition by phenylalanine and BCH as well as glutamine andserine. In lithium buffer, glutamine strongly inhibited anti[14C]FACBC uptake in tumor cells as well as T/B cells, but thisis not due to transport via system N as mentioned above. These
results suggest that systems ASC and L are the main AATs in
Na+-dependent and Na+-independent anti-[14C]FACBC transport, respectively.
System L is thought to be the main AAT for [14C]Mettransport in MLLB2 and C6 cells, for the same reasons asmentioned above.

Table 2. Uptake ratio of activated inﬂammatory cells to tumor cells (with D-glucose)
Activated inﬂammatory cells

T cells
B cells

These ratios were calculated from Fig. 2a–d

pH

6.0
7.0
8.0
6.0
7.0
8.0

Anti-[14C]FACBC[14C]Met

vs. MLLB2

vs. C6

vs. MLLB2

vs. C6

0.5
0.6
0.6
0.4
0.4
0.5

0.5
0.5
0.5
0.4
0.4
0.4

0.1
0.1
0.2
0.1
0.1
0.1

0.1
0.1
0.1
0.1
0.1
0.1

S. Oka et al.: Anti-[18F]FACBC Transport Mechanisms in Inflammatory and Cancer Cells

Fig. 3. Competitive inhibition of anti-[14C]FACBC and[14C]Met transport in rat activated T cells and B cells, andPCa (MLLB2) and glioma (C6) cell lines in sodium (a), choline(b), and lithium (c) buffer (n=5–21). The intensity of thecalculated inhibitory effect was categorized into five classes
as indicated.

Discussion
Here, we compared the uptake of anti-[14C]FACBC, [14C]Met,and [14C]FDG in rat inﬂammatory cells (T cells, B cells,
granulocytes, macrophages) and tumor cell lines (MLLB2 andC6) by time-course experiments. We found that the uptake oftracers used in this study was enhanced with inﬂammatory cell
activation, and relatively high inﬂammatory cell to tumor cell anti[14C]FACBC uptake ratios in T/B cells, comparable to or higher
than those of [14C]FDG. Next, we elucidated differences in anti[14C]FACBC and [14C]Met transport mechanisms between

327

inﬂammatory and tumor cells through a series of pH dependency
and competitive inhibition uptake experiments. We demonstrated
that (1) Na+-dependent (especially system ASC) and Na+independent (especially system L) AATs are likely the main routeof anti-[14C]FACBC and [14C]Met uptake, respectively, in T/Bcells; (2) in cancer cell lines, the contribution of both carriersystems to anti-[14C]FACBC transport was 50:50 and this ratio is
pH-dependent; and (3) Na+-independent AATs (especially systemL) were predominant in [14C]Met uptake in cancer cells.
Generally, AA PET tracers such as [11C]Met, O-18Fﬂuoroethyl-L-tyrosine, and O-18F-ﬂuoromethyl-L-tyrosine arethought to be preferable for the differentiation of tumor andinﬂammatory tissues, because they are taken up substantially bycancer cells but not inﬂammatory cells in comparison to[18F]FDG [14, 15]. We previously showed an apparently higher
ratio of tumor-to-inﬂammation accumulation for anti[18F]FACBC than [18F]FDG using a rat PCa and lymphadenitis
model in vivo [5]. However, recent pre-clinical and clinicalstudies revealed [11C]Met uptake in inﬂamed regions [15, 16].
Moreover, some false-positive uptake, possibly by the accumulation of anti-[18F]FACBC in inﬂammatory regions such as
prostatitis and benign prostatic hyperplasia, has been reported in
PCa patients [7]. In fact, we show relatively low tumor-toinﬂammatory cell anti-[14C]FACBC uptake ratios (especiallyagainst activated T/B cells), though much higher tumor-toinﬂammatory cell uptake ratios were observed with macrophages and granulocytes.
Anti-tumor responses by inﬂammatory cells and tumorpromoting inﬂammation coexist in the tumor microenvironment, and an inﬂammatory microenvironment is essential for
all tumors [17]. Inﬂammatory cells in the tumor microenvironment serve as sources of cytokines and chemokines, which areessential for establishing cancer implant viability [17]. It isestimated that 80 % of PCa patients have some degree ofinﬂammation in the prostate [8]. Although several kinds ofinﬂammatory cells such as T/B cells, neutrophils, macrophages, dendritic cells, and mast cells are observed in the tumor
microenvironment [17], lymphocytes are most frequently
observed in PCa, and macrophages and neutrophils to a lesser
extent [8]. These inﬂammatory cells are detected even in
normal prostate and benign prostatic hyperplasia; 70–80 % as T
cells and 10–15 % as B cells, with approximately 40 % of theintra-prostatic T cells activated [8].
AAT expression in inﬂammatory cells has been reported.
For example, T and/or B cells show cell-surface expressionof systems ASC (ASCT1, ASCT2), L (LAT1), and A(SNAT1, SNAT2), but not XC− (xCT) [18–21]; these levels
increase on activation [22–24]. Among these, systems A,
ASC, and L are the main neutral AA transport systems in
mammalian cells, and are over-expressed in various cancers
including PCa [25]. Consequently, the transport routes ofnaturally occurring AAs in activated inﬂammatory cells andcancerous cells are thought to be similar. As discussed above,
systems ASC and L would be the main carriers for anti[18F]FACBC transport in activated T cells and cancer cells,
although their contributions would vary somewhat with

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S. Oka et al.: Anti-[18F]FACBC Transport Mechanisms in Inflammatory and Cancer Cells

extracellular pH changes. Moreover, we have reported that anti[14C]FACBC uptake into cells is likely similar to that of Lglutamine, because L-glutamine is the most effective anti[14C]FACBC uptake inhibitor in the presence of Na+, and the
afﬁnities (Km) of anti-[14C]FACBC and L-glutamine for ASCT2
were similar (92 and 24–90 μM, respectively) [10, 11]. LGlutamine is the most abundant AA in blood and is thought to be
as important as glucose as fuel for cancer cells as well as
inﬂammatory cells; it is essential for inﬂammatory cell functions
such as T cell proliferation, B cell differentiation, macrophage
phagocytosis and antigen-presentation, and neutrophil superoxide production [26]. We therefore concluded that the relatively
high anti-[18F]FACBC accumulation in tumor cells and in T/Bcells is caused by its preference for system ASC, and some falsepositives caused by anti-[18F]FACBC accumulation in inﬂamed
regions are inevitable in FACBC-PET of prostate and othercancer patients [7].
Recently, glutamine-based PET tracers such as L-[5-11C]glutamine and 18F-(2S,4R)4-ﬂuoroglutamine have been reported to clearly visualize glioma and mammary tumors in rodent
models [27, 28]. Because the uptake mechanisms of thesetracers are likely to be more speciﬁc for ASCT2 than that ofanti-[18F]FACBC, the process of glutamine-based tracer
uptake by inﬂammatory cells would be interesting.
FDG-PET may exhibit false-positive uptake caused by[18F]FDG accumulation at lesions such as abscesses andgranulomatous areas, including activated inﬂammatory cells
in cancer patients [29]. For example, glioma patients exhibit
signiﬁcant neutrophil inﬁltration, the degree of which correlated well with the tumor malignancy grade [30]. Kaim et al. [31]showed that several neutrophils inﬁltrated into abscesses
containing central necrosis, and the increased [18F]FDG
accumulation corresponded to granulocyte inﬁltration in a rat
abscess model. Furthermore, [11C]Met, but not [18F]FDG, hasbeen shown to be useful for differentiating cancerous lesions
from granulomatous reactions [32] or radiation necrosis afterradiotherapy for glioma [33]. Here, anti-[14C]FACBC and[14C]Met uptake was relatively high in activated T and/or B
cells, but low in activated granulocytes and macrophages
compared to tumor cells. Taken together, FACBC-PET may
have a lower false-positive uptake than FDG-PET by improving tumor delineation in patients with acutely inﬂamed lesions,though it may be disadvantageous with chronic inﬂammation.
Using rat inﬂammatory cells, we have demonstrated thepossibility that anti-[18F]FACBC accumulates in inﬂammation, especially in activated T/B cells, to a greater extent thanpreviously believed. However, species differences between
humans and rodents have been disputed in pharmacokinetics.
In fact, it has been reported that some AATs, such as LAT1 andASCT2, show lower expression at the blood–brain barrier inhumans than in mice [34]. Species differences in AAT
expression between human and rat inﬂammatory cells
might exist; thus, we recognize the critical importance ofanti-[18F]FACBC transport studies in human inﬂammatory
cells. Moreover, further investigation of more complex invivo conditions and in the tumor microenvironment iswarranted to understand the possibility of false-positive
uptake and the usefulness of anti-[18F]FACBC imaging
for cancer patients.

Conclusion
Our study suggests that signiﬁcant uptake of anti-[18F]FACBC
into activated T/B cells is inevitable and might cause falsepositives in FACBC-PET imaging of cancer patients. However, anti-[18F]FACBC is expected to have less false-positives
compared with FDG-PET, because of its low uptake into
granulocytes and macrophages. Furthermore, the central player
for anti-[18F]FACBC transport in T/B cells is system ASC,
while that for [11C]Met is system L. Both systems are important
for anti-[18F]FACBC uptake in tumor cells, but system L
is responsible for [11C]Met uptake. Therefore, the relatively
high anti-[18F]FACBC accumulation not only in tumor cells
but also in T/B cells is likely caused by its preference forsystem ASC.
Acknowledgments. The authors thank Mr. Shiro Yoshida and Ms. Sachiko
Naito for their assistance in animal treatments and cell cultures, respectively.
Conﬂict of interest. Shuntaro Oka, Hiroyuki Okudaira, Masahiro Ono, andYoshifumi Shirakami are employees of Nihon Medi-Physics Co. Ltd. Mark
M. Goodman and Emory University have patent rights for anti-[18F]FACBC
and are eligible to receive royalties on anti-[18F]FACBC from Nihon MediPhysics Co. Ltd. Mark M. Goodman, David M. Schuster, and Keiichi Kawai
have ongoing research collaborations with Nihon Medi-Physics Co. Ltd.

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